1. Field of the Invention
The present invention relates to detection techniques in bio-analysis, particularly optical detection in bio-separation through a separation column, and more particularly detection of emissions from radiation excitations in capillary electrophoresis.
2. Description of Related Art
Bioanalysis, such as DNA analysis, is rapidly making the transition from a purely scientific quest for accuracy to a routine procedure with increased, proven dependability. Medical researchers, pharmacologists, and forensic investigators all use DNA analysis in the pursuit of their tasks. Yet due to the complexity of the equipment that detects and measures DNA samples and the difficulty in preparing the samples, the existing DNA analysis procedures are often time-consuming and expensive. It is therefore desirable to reduce the size, number of parts, and cost of equipment, to make easy sample handling during the process, and in general, to have a simplified, low cost, high sensitivity detector.
One type of DNA analysis instruments separates DNA molecules by relying on electrophoresis. Electrophoresis techniques could be used to separate fragments of DNA for genotyping applications, including human identity testing, expression analysis, pathogen detection, mutation detection, and pharmacogenetics studies. The term electrophoresis refers to the movement of a charged molecule under the influence of an electric field. Electrophoresis can be used to separate molecules that have equivalent charge-to-mass ratios but different masses. DNA fragments are one example of such molecules.
There are a variety of commercially available instruments applying electrophoresis to analyze DNA samples. One such type is a multi-lane slab gel electrophoresis instrument, which as the name suggests, uses a slab of gel on which DNA samples are placed. Electric charges are applied across the gel slab, which cause the DNA sample to be separated into DNA fragments of different masses.
Another type of electrophoresis instruments is the capillary electrophoresis (CE) instrument. By applying electrophoresis in a fused silica capillary column carrying a buffer solution, the sample size requirement is significantly smaller and the speed of separation and resolution can be increased multiple times compared to the slab gel-electrophoresis method. These DNA fragments in CE are often detected by directing light through the capillary wall, at the components separating from the sample that has been tagged with a fluorescence material, and detecting the fluorescence emissions induced by the incident light. The intensities of the emission are representative of the concentration, amount and/or size of the components of the sample. In the past, Laser-induced fluorescence (LIF) detection methods had been developed for CE instruments. Fluorescence detection are often the detection method of choice in the fields of genomics and proteomics because of its outstanding sensitivity compared to other detection methods.
Some of the challenges in designing CE-based instruments and CE analysis protocols relates to sample detection techniques. In the case of fluorescence detection, considerable design considerations had been given to, for example, radiation source, optical detection, sensitivity and reliability of the detection, cost and reliability of the structure of the detection optics. In the past, relatively high power light source is required, such as Laser systems. When light is directed through the capillary wall at the separated sample components in the capillary bore, light scatters at the outside capillary wall/air interface and the inside capillary wall/buffer interface (Raman scattering), which obscures or corrupts the fluorescence emission intensity. Similarly, fluorescence emissions scatter at the wall interfaces. In the past, various techniques were developed for more completely collecting the fluorescence emissions to improve signal intensity and hence detection sensitivity. These techniques involve additional moving and non-moving components that add to the relative complexity and cost of the detection setup.
The design limitations of prior art electrophoresis instruments are exacerbated in the development of multi-capillary CE-based instruments. For example, confocal scanning laser induced fluorescence (LIF) detection has been adopted in multi-capillary electrophoresis systems. The scanning confocal detection relies on a scanning optical system. The use of moving parts is not ideal when taking simplicity, robustness, and lower cost of the instrument into consideration. Also, the shallow focal depth of the microscope objective for the confocal detector puts severe demands on the mechanical and optical component tolerances. Further, the optical scanning method generally involves a longer duty cycle per capillary. Thus, should the instrument be scaled up in order to generate higher throughput, the sensitivity of the system may be impaired.
The present invention provides a simplified, low cost, efficient, highly sensitive, non-moving and stable micro-optical detection configuration for bio-separation (e.g., capillary electrophoresis) through a separation column having a separation channel filled with a separation support medium (e.g., a liquid or sieving gel including a running buffer). More particularly, the present invention is directed to an improved detection configuration for the detection of radiation emitted by sample analytes (e.g., fluorescence emission), in contrast to radiation absorbance detection techniques.
In one aspect of the present invention, the zone for optical detection of sample components is located at a widened zone along the separation channel. In one embodiment of the present invention, the widened detection zone is a micro-bore collar having a micro-channel that coaxially surrounds the exit of a capillary column that defines a capillary channel. A separation support medium (e.g., a liquid or sieving gel) including a running buffer fills the capillary column and the collar.
According to another embodiment of the present invention, excitation radiation is directed at the detection zone from outside the walls of the widened detection zone, with on-column or off-column optical detection.
In another aspect of the present invention, incident radiation (e.g., from a laser or LED source) for the detection is directed at the detection zone axially (i.e., in the direction of the axis, but does not necessarily have to be along the axis) along the separation medium, instead of through the boundary walls of the detection zone or the separation column. In one embodiment, incident radiation is directed via an optic fiber (hereinafter referred to as an excitation fiber) that extends axially along the separation medium to the proximity of the detection zone. Emitted or output radiation from the detection zone passes through the boundary walls about the detection zone for detection (i.e., off-column detection). According to one embodiment of off-column detection, a curved reflective collector is used to better capture and collect the emitted radiation, for example by using a parabolic, ellipsoidal, toroidal, or spherical reflector as a light collector. According to another embodiment of off-column detection, high collection angle micro-lenses are used to facilitate in capturing the maximum amount of emitted radiation.
According to another embodiment of the present invention, at least two excitation fibers direct incident radiation to provide incident radiation at different wavelengths.
According to a further embodiment of the present invention, at least two radiation sources direct radiation at different wavelengths to the detection zone via a single excitation fiber (e.g., by means of a dichroic beam combiner).
According to another embodiment of the present invention, at least two lasers and a beam combiner are combined with an excitation fiber, which is incorporated inside a micro-channel.
In a further aspect of the present invention, emitted radiation signals representative of the sample components are collected from the detection zone axially along the separation medium (hereinafter referred as on-column detection), instead of through the boundary walls of the detection zone or the separation column (which is off-column detection). In one embodiment, emitted signals are collected via an optic fiber (hereinafter referred to as the emission fiber) that extends from the proximity of the detection zone along the separation medium.
According to another embodiment of the present invention, two fibers (an excitation fiber and an emission fiber) are incorporated into detection collar, one for excitation radiation and the other for emitted radiation detection.
According to a further embodiment of the present invention, confocal radiation detection optics is configured to make use of a single dual-purpose (excitation and emission) fiber to direct incident radiation at the detection zone and emitted radiation from the detection zone to a detector. An optical element (e.g., a beam splitter such as a dichroic beam combiner) is employed to direct incident radiation from a source at the detection zone through the single dual-purpose fiber, and to separate the emitted radiation from the detection zone arriving through with the same dual-purpose fiber.
Various combinations of the foregoing embodiments of detection configurations may be implemented without departing from the scope and spirit of the present invention. For example, using a combination of dual-purpose fiber, excitation fiber and/or emission fiber, a combination of on-column and off-column detection at different wavelengths of incident radiations may be configured.
In a further aspect of the present invention, the optical detection of the present invention may be scaled up and implemented in a multi-channel CE system that comprises multiple capillary separation channels, using similar axial incident radiation and/or emitted radiation detection configurations set forth above.